Cyclotriphosphazatriene-derivatives as soil urease activity inhibitors

ABSTRACT

Test show that 2,2,4,4,6,6-hexaaminocyclotriphosphazatriene, 2-phenoxy-2,4,4,6,6-pentaaminocyclotriphosphazatriene, 2,4-diphenoxy-2,4,6,6-tetraaminocyclotriphosphazatriene and 2,4,6-triphenoxy-2,4,6-triaminocyclotriphosphazatriene (also frequently called phosphonitrilic derivatives) of the formula ##STR1## are highly effective inhibitors of urease activity in agricultural soil systems wherein 
     (1) R 1  . . . R 3  &#39;=NH 2  or 
     (2) R 1  &#39;=R 2  &#39;=R 3  &#39;=R 2  =R 3  =NH 2  and R 1  =OC 6  H 5  or 
     (3) R 1  &#39;=R 2  &#39;=R 3  &#39;=R 3  =NH 2  and R 1  =R 2  =OC 6  H 5  or 
     (4) R 1  &#39;=R 2  &#39;=R 3  &#39;=NH 2  and R 1  =R 2  =R 3  =OC 6  H 5 .

The invention herein described may be manufactured and used by or forthe Government for governmental purposes without the payment to us ofany royalty therefor.

This application is a division of application Ser. No. 688,101, filedDec. 31, 1984, now U.S. Pat. No. 4,618,691, 10/21/86, which in turn is acontinuation of parent application Ser. No. 625,424, filed 7-2-85 nowDefensive Publication No. T105,605, published July 2, 1985.

INTRODUCTION

The enzyme urease (urea amidohydrolase, EC 3.5.1.5) is a ubiquitouscomponent of many soil systems and has been isolated from a number ofmicrobes and many different plants. In soil systems, urease activitiesserve to catalyze the hydrolysis of urea to produce ammonia and carbondioxide according to the reaction: ##STR2## The ammonia produced issubsequently hydrolyzed to nutrient ammonium salts.

    NH.sub.3 +H.sub.2 O⃡NH.sub.4.sup.+ +OH.sup.-

The NH₄ ⁺ is then transformed to NO₃ ⁻ by aerobic nitrifying bacteria inthe soil.

    NH.sub.4.sup.+ +2O.sub.2 →NO.sub.3.sup.- +H.sub.2 O+2H.sup.+

This sequence of reactions serves a vital function in providinginorganic nitrogen for growing plants. However, urease-inducedhydrolysis of urea can cause a considerable loss of volatile ammonia,especially when urea fertilizers are surface applied to agriculturalsoils [Darrell W. Nelson, Nitrogen in Agricultural Soils, Am. Soc.Agron., Madison, WI, p. 327-358 (1982)]. Most of ammonia volatilizationfrom urea occurs in the first week after application. Moderate delays inurea hydrolysis during this time period can greatly reduce ammoniavolatilization losses for several reasons. For instance, the farmer hasmore time to incorporate urea beneath the soil surface before suchammonia losses occur. There is a greater probability of receiving rainwith resulting incipient percolation of fertilizer nitrogen values intothe soil before such ammonia losses occur. Also, a larger fraction ofthe applied nitrogen is converted to NO₃ ⁻ before being lost as ammonia.

Urea and urea-containing fertilizers presently account for about 30percent of the fertilizer nitrogen applied in the United States [J.Darwin Bridges, Fertilizer Trends 1982, TVA (1983)], and urea accountsfor as much as 60 percent of the fertilizer nitrogen applied worldwide(unpublished TVA data). The trend-line prediction for these percentagesis for an increase because urea has a high nitrogen content, lowtransportation cost, and low production cost relative to alternativenitrogen sources, such as ammonium nitrate and ammonium sulfate.Inasmuch as the relative importance of urea as a primary nitrogenfertilizer is expected to increase to even greater proportions than itnow enjoys and substantial amounts of such urea and/or urea-containingfertilizers are applied in situations, such as reduced tillage,pastures, and nonmechanized agriculture, where it is impractical tomechanically incorporate urea to prevent ammonia volatilization, thedevelopment of suitable urease inhibitors is an endeavor of considerableimportance for both domestic and international agriculturalconsiderations.

Considerable effort is being devoted by a number of research groups inboth the private and the public sector to develop suitable ureaseinhibitors. A particularly promising class of urease inhibitors iscompounds containing phosphoroamide groups, R_(x) PO(NH₂)₃.sbsb.-x,where R=NH₂, OH, phenol, etc. Several researchers in the art havedemonstrated that phenyl phosphorodiamidate, (C₆ H₅ O)PO(NH₂)₂, is anextremely potent inhibitor of urease activity [P. Held, S. Lang, E.Tradler, M. Klepel, D. Drohne, H. J. Hartbrich, G. Rothe, H. Scheler, S.Grundmeier, and A. Trautmann, East German Pat. No. 122,177 (Cl.C05G3/08, Sept. 20, 1976), Chem. Abstr. 87:67315W; D. A. Martins and J.M. Bremner, Soil Sci. Soc. Am. J. 48:302-305 (1984)]. Recently Baylessand Millner [U.S. Pat. No. 4,242,325 (1980) and U.S. Pat. No. 4,182,881(1980)] showed that phosphoryltriamide, PO(NH₂)₃ and a series ofN-[diaminophosphinyl]arylcarboxamides are also powerful ureaseinhibitors. Other investigators have shown that diamidophosphoric acid,PO(NH₂)₂ OH, and monoamidophosphoric acid, PO(NH₂)(OH)₂, are alsoeffective urease inhibitors [A. Barth, W. Rollka, and H. J. Michel,Wissenschaftliche Beitraege-Martin Luther Universitaet Halle Wittenberg,No. 2, 5-10 (1980); N. E. Dixon, C. Gazzola, J. J. Waters, R. L.Blakeley, and B. Zerner, J. Am. Chem. Soc. 97:4131 (1975)].

The present invention relates to the discovery that certain materialsmay be effectively utilized as potent urease activity inhibitors inagricultural soil systems including cyclotriphosphazatriene-derivativesof the formula ##STR3## wherein (1) R₁ . . . R₃ '=NH₂ or

(2) R₁ '=R₂ '=R₃ '=R₂ =R₃ =NH₂ and R₁ =OC₆ H₅ or

(3) R₁ '=R₂ '=R₃ '=R₃ =NH₂ and R₁ =R₂ =OC₆ H₅ or

(4) R₁ '=R₂ '=R₃ '=NH₂ and R₁ =R₂ =R₃ =OC₆ H₅

SUMMARY OF THE INVENTION

In arriving at the gist underlying the concept of the instant invention,it was conceived that cyclotriphosphazatriene-derivatives, even thoughthey are not members of the phosphoroamide class of compounds discussedabove, should also be investigated as urease activity inhibitors.Although many research groups, especially R. A. Shaw et al. [R. A. Shaw,Phosphorus and Sulfur 4:101-121 (1978)], have concerned themselves withthe preparation of cyclotriphosphazatriene-derivatives, the threephenoxyaminocyclotriphosphazatrienes presented above are thought to beactually new compounds, reported now for the first time, because noinformation about their preparation and chemistry has been found. Of thecompounds of interest, only the2,2,4,4,6,6-hexaaminocyclotriphosphazatriene (phosphonitrilic hexaamide)has been reported. The aqueous solution hydrolysis of2,2,4,4,6,6-hexaaminocyclotriphosphazatriene was studied by Dostal,Kouril, and Novak [K. Dostal, M. Kouril, and J. Novak (J. E. PurkyneUniv., Brno, Czech.) Z. Chem. 4(9):353 (1964), (Chem. Abstr.62:4670,g)]. They report that the aqueous solution hydrolysis of2,2,4,4,6,6-hexaaminocyclotriphosphazatriene proceeds by the followingsequence of reaction steps: ##STR4## Hence, the hydrolysis of2,2,4,4,6,6-hexaaminocyclotriphosphazatriene produces phosphoryltriamideand a consecutive series of ammonium salts of phosphoroamide compounds,all of which have been demonstrated supra to be urease activityinhibitors.

In this in vitro experiment (in an enzyme and soil free system) thereaction temperature employed by Dostal, Kouril, and Novak was notreported; however, similar studies conducted in TVA laboratory testsrequired a minimum temperature of 70° C. in order to obtain convenientlymeasurable reaction rates (unpublished data, TVA, 1974).

These results are also in agreement with the publication of W. Topelmannand coworkers [W. Topelmann, H. Kroschwitz, D. Schroter, D. Patzig, andH. A. Lehmann, Z. Chem. 19:273-380 (1979)], where the hydrolysis of2,2,4,4,6,6-hexaaminocyclotriphosphazatriene was conducted by pH 8 andtemperatures of 15° C. and 30° C., hydrolyzing very slowly in about 300and 100 hours, respectively. They demonstrated also, that not onlyphosphoryltriamide and phosphoryldiamide were produced during thehydrolysis but also different imidoamidopolyphosphates in significantamounts, which could also have urease inhibitory properties.

An investigation by Dick and Tabatabai [W. A. Dick and M. A. Tabatabai,Geoderma 21:175-182 (1978)] showed that hexaaminocyclotriphosphazatrienehydrolyzed very slowly (6-13 percent hydrolyzed in 7 days) in threedifferent soil systems at 20° C.

Although the literature teaches that2,2,4,4,6,6-hexaaminocyclotriphosphazatriene exhibits characteristicssuch that it appears to resist hydrolysis both in vitro and in vivo,i.e., in laboratory solutions at or near room temperature and in manysoil systems during spring or early summer applications, respectively.We have unexpectedly found that hexaaminocyclotriphosphazatriene and thephenoxyaminocyclotriphosphazatrienes are excellent urease inhibitors.

Some nitrogen-containing heterocyclic compounds such as triazolederivatives or a N-ethylmaleinimide having structures similar to thecyclotriphosphazatrienes have been reported to be effective ureaseinhibitors in experiments with the isolated enzyme [P. Mildner and B.Mihanovic, Croat. Chem. Acta 46:79-82 (1974)] and also in soilexperiments, with the herbicide 3-amino-1,2,4-triazole [S. M. Gauthier,S. S. Ashtakala, and J. A. Lenoir, Hort. Science 11:481-482 (1976)]. Itmay be possible that the two inhibitor classes have substantially thesame mechanism of inhibition, to wit, reacting with the essentialsulfhydryl group(s) on the active site(s) of the urease. At this time,however, we can only speculate that the inhibitory properties of thecyclotriphosphazatriene derivatives result either from some yetunidentified chemical properties and/or characteristics of the compoundsthemselves. If the mechanism is related to reacting with, or inhibitingof such sulfhydryl group(s), it might be classified as irreversibleinhibition but more probably as competitive inhibition.

Several hundred scientific papers have been published on urease sinceSumner (1926) first produced the classical octahedral crystals andshowed that the enzyme was a protein, but it was in 1969 that Zerner'sgroup [R. L. Blakeley, E. C. Webb, and B. Zerner, Biochemistry8:1984-1990 (1969)] prepared a highly purified urease with a fullspecific activity and in at least 99% a homogeneous state. Theyestablished with this preparation a reproducible molecular weight (about590,000) and proposed that the molecule contained six subunits withasparagine as the N-terminal amino acid. Although previous work [J. F.Ambrose, G. B. Kistiakowsky, and A. G. Kridl, J. Amer. Chem. Soc.73:1232 (1951)] had indicated that four or eight essential SH-groupswere involved in the urea-hydrolysis reaction, Zerner's group could onlyconfirm that the active site SH-groups "react slowly withN-ethylmaleimide," but they were unable to define unequivocally thenumber of "essential SH groups" in the 590,000 molecular weight species.In addition, Kobashi et al. [K. Kobashi, J. Hase, and T. Komai, Biochem.Biophys. Res. Commun. 23:34 (1966)] on the basis of inhibition byhydroxamic acids suggested that the number of active sites in the590,000 molecular weight species of sword bean urease was 2. Theseresults seem to be confirmed by the discovery that highly purifiedurease from jack bean [N. E. Dixon, C. Gazzola, R. L. Blakeley, and B.Zerner, J. Am. Chem. Soc. 97:4131 (1975)] and from tobacco, rice, andsoybean [J. C. Polacco, Plant Science Letters 10:249-255 (1977)]contained stoichiometric amounts of nickel (2 atoms per active site),demonstrating simultaneously the first biological role definitelyassigned to nickel. Over the last few years considerable effort has beenmade to elucidate the mechanism of the urease reaction. Althoughattempts to demonstrate the formation of a carbamoyl-enzymeintermediate, which was postulated many years ago, have so far failed,Zerner's group [N. E. Dixon, P. W. Riddles, C. Gazzola, R. L. Blakeley,and B. Zerner, Can. J. Biochem. 58:1335-1344 (1980)] proposed amechanism of reaction on the base of a carbamoyl-transfer reaction andwhere the substrate is activated toward nucleophilic attack byO-coordination to a Ni².spsp.+ ion. Both Ni².spsp.+ ions are involved inthis proposed mechanism. A second mechanism of reaction based on thedetermination of kinetic isotope effects [R. Medina, T. Olleros, andH.-L. Schmidt, IN Proc. 4th Int. Conference on Stable Isotopes, p.77-82, H.-L. Schmidt, H. Forstel, K. Heizinger (Eds.), Julich, March1981, Elsevier, Amsterdam (1982)] was proposed. These results indicatedthe existence of an enzyme bound carbamate intermediate and demonstratedthat the enzyme-Ni-substrate complex decomposes releasing the first NH₃in a slow, rate-limiting step.

An additional complication develops from the tendency of the urease toform polymers and isozymes changing the properties of the originalmonomeric enzyme and probably the mechanism of reaction [W. N. Fishbeinand K. Nagarajan, Arch. Biochem. Biophys. 144:700-714 (1971)]. Finallythe properties of soil urease differ significantly from those of ureasesfrom other sources [J. M. Bremner and R. L. Mulvaney, IN Soil Enzymes,p. 149-196, R. G. Burns (Ed.), Academic Press (1978)], and it is muchmore difficult to obtain reliable kinetic data for enzymes inheterogeneous environments, such as soil, than for enzymes inhomogeneous solutions.

While many urease inhibitors have been identified, few kineticdescriptions include the type of inhibition. The reversible andcompetitive inhibition of sword bean urease by a wide variety ofhydroxamic acids was discovered by Kobashi et al. [K. Kobashi, J. Hase,K. Uehara, Biochim. Biophys. Acta 65:380-383 (1962)]. Kinetic andspectral studies performed by B. Zerner and coworkers [N. E. Dixon, J.A. Hinds, A. K. Fihelly, C. Gazzola, D. J. Winzor, R. L. Blakeley, andB. Zerner, Cand. J. Biochem. 58:1323-1334 (1980)] established thathydroxamic acids were reversibly bound to active-site nickel ions injack bean urease. Chemical and physical studies of the enzymaticallyinactive phosphoramidate-urease complex provide convincing evidence thatphosphoramidate binds reversibly to the active-site nickel ion [N. E.Dixon, R. L. Blakeley, and B. Zerner, Can. J. Biochem. 58:481-488(1980)].

The kinetics of urease inhibition by phenyl phosphorodiamidate whichdemonstrates a competitive inhibition and hydroquinone which exemplifiesa mixed inhibition mechanism were performed by L. J. Youngdahl and E. R.Austin at IFDC (unpublished results). A kinetic study of the soil ureaseinhibition by six substituted ureas, compounds which are used asherbicides, showed that all six compounds exhibited mixed inhibitioncharacteristics (competitive and noncompetitive) [S. Cervelli, P.Nannipieri, G. Giovannini, and A. Perna, Pesticide Biochem. Physiol.5:221-225 (1975)].

There are very few additional publications on kinetic studies concerningsoil ureases [J. M. Bremner and R. L. Mulvaney, IN Soil Enzymes, p.149-196. R. G. Burns (Ed.), (1978)]. The main work in this area has beento establish the inhibitory properties of potential test compounds,irrespective of the kind of inhibition that is responsible for theretardation of the urea hydrolysis. However, the successful use of thistechnology by the fertilizer industry does not require that themechanism be identified.

Taking into consideration all of this information one can establish thateven though urease has been extensively studied for about 60 years, themechanism of action and the mechanism of inhibition of this enzyme,especially in heterogeneous environments such as soils are, at best,only partially known.

OBJECT OF THE INVENTION

The principal object of the present invention is to identify andcharacterize a group of highly effective inhibitors which will, whenadmixed with urea or urea-containing fertilizers, prevent or greatlyreduce the loss of ammoniacal nitrogen from agricultural soils resultingfrom the urease-induced hydrolysis urea.

While cyclotriphosphazatriene-derivatives have now been identified aseffective inhibitors for the purpose of the present invention, it is nowpostulated in view of information gleaned by reducing the presentinvention to practice that perhaps many derivatives of the abovecompounds which contain the PN cyclic structure, especially thecyclotetraphosphazatetraene derivatives, are also potentially effectiveurease inhibitors. For example, the following compounds having the sameskeleton structure of cyclic PN but with the R groups replaced withother radicals or elements, such as: ##STR5## where R₁ to R₄ ' may behydrogen or the following functional groups: halogen, pseudohalogen,hydroxy, sulfhydryl, alkyl or substituted alkyl, alkenyl or substitutedalkenyl, alkynyl or substituted alkynyl, aryl or substituted aryl, aminoor substituted amino, hydrazino or substituted hydrazino, acyl orsubstituted acyl, aroyl or substituted aroyl, alkoxy or substitutedalkoxy, aryloxy or substituted aryloxy, thioalkoxy or substitutedthioalkoxy, thioaryloxy or substituted thioaryloxy, anilino orsubstituted anilino, heterocyclic or substituted heterocyclic, as wellas combinations of the above, etc., are likely to be effective ureaseinhibitors. A further opportunity to capitalize on our observations todate may be effected by the use of the parent materials, supra,polymerized to the appropriate degree to yield the optimum combinationof solubility and inhibitory effect upon the urease enzyme.

It will now, of course, be appreciated that the substitution suggestedsupra for the R₁ to R₄ ' groups on the original skeleton structure couldlead those skilled in the art to the testing and investigation of greatmultiplicity of compounds. We have been particularly interested ininvestigating the characteristics of the following three derivatives, towit, P₃ N₃ [N(CH₃)₂ ]₆,2,2,4,4,6,6-hexa(dimethylamino)cyclotriphosphazatriene; P₃ N₃ (NHCH₃)₆,2,2,4,4,6,6-hexa(monomethylamino)cyclotriphosphazatriene and P₃ N₃(NH₂)₂ [N(CH₃)₂ ]₄,2,2,4,4,-tetra(dimethylamino)-6,6-diaminocyclotriphosphazatriene. Testresults of these three materials as well as others as set forth inExample III, infra, show that these materials do not exhibit ureaseinhibition.

Similar soil tests performed with P₃ N₃ (Cl)₅ (OC₆ H₅),2-phenoxy-2,4,4,6,6-pentachlorocyclotriphosphazatriene; P₃ N₃ (Cl)₄ (OC₆H₅)₂, 2,4-diphenoxy-2,4,6,6, tetrachlorocyclotriphosphazatriene; and P₃N₃ (Cl)₃ (OC₆ H₅)₃,2,4,6-triphenoxy-2,4,6-trichlorocyclotriphosphazatriene, showed thatthese compounds also are not effective urease inhibitors.

DESCRIPTION OF THE DRAWINGS

The present invention, together with further objects and advantagesthereof, will be better understood from a consideration of the followingdescription taken in connection with the accompanying drawing in which:

FIG. 1 is a graphical illustration of urease activity inhibition as afunction of time.

FIG. 2 shows the change in percent urease inhibition for times up to 21days obtained using a modification of the evaluation procedure thatpermits measurement of urease inhibition in a system that is independentof time of urea addition as discussed infra, page 18, lines 14-18.

FIG. 3 and FIG. 4 show the calculated percent urease inhibition that canbe obtained using physical mixtures of some of the test compounds. Forthe sake of convenience and a better understanding, FIGS. 2 to 4 arediscussed in detail, in Example VI infra.

Referring more specifically to FIG. 1, the results presented thereinwere plotted and extrapolated on the basis of the data contained atleast in part in Table V and using J. M. Bremmer's concept of thepercentage urease inhibition* [L. A. Douglas and J. M. Bremner, SoilBiol. Biochem., 3:309-315 (1971)]. FIG. 1 shows the different degrees ofinhibition of the cyclotriphosphazatrienes compared to PPDA, which has ahigher initial percentage of urease inhibition but decreases to 0% inless than 9 days. The 2-phenoxy-2,4,4,6,6pentaaminocyclotriphosphazatriene and 2,4-diphenoxy-2,4,6,6tetraaminocyclotriphosphazatriene show a slightly longer period ofurease inhibition but also approach 0% inhibition in about 10 days.

DESCRIPTION OF PREFERRED EMBODIMENTS

For ease and convenience of application, the cyclotriphosphazatrienederivatives may be incorporated into urea or urea-containing fertilizersby: mixing, prilling, granulating, coating, or other means familiar tothose knowledgeable in the art of producing and/or blending fertilizermaterials.

Examples I, II, IV, V, and VI, infra, are in the nature of positiveexamples and depict the rather significant effect that2,2,4,4,6,6-hexaaminocyclotriphosphazatriene,2-phenoxy-2,4,4,6,6-pentaaminocyclotriphosphazatriene,2,4-diphenoxy-2,4,6,6-tetraaminocyclotriphosphazatriene, and2,4,6-triphenoxy-2,4,6-triaminocyclotriphosphazatriene exhibit as soilurease activity inhibitors.

Example III, infra, is offered in the manner of a negative example andshows various compounds, including the derivative mentioned supra, thatdo not act or display characteristics having utility as soil ureaseactivity inhibitors.

In order that those skilled in the art can better understand andappreciate the work reported herein, brief descriptions, infra, of thetesting methods employed are given before the specifics of Examples I,II, III, IV, V, and VI are discussed.

Testing Methods

Urease activity inhibitor test compounds may be evaluated either inaqueous or in soil systems. When aqueous systems are used, urea plus atest compound with possible urease inhibition activity and relativelypure urease enzyme are incubated together to determine the effects ofthe test compound on urease-catalyzed hydrolysis of urea. When soilsystems are used, urea and the test compound are added to moist soil,and the urease enzyme is supplied from the soil. The main disadvantageof using soil systems is that the true activity of test compounds may bemasked because of reactions between the test compound and soil. Thus,basic studies for understanding chemical structure-activityrelationships are usually done in aqueous systems. However, soil systemsmust be used to determine the principal applicability of test compoundssince soil can significantly modify inhibitory effects of thesecompounds.

The most common and conventional method for evaluating potential ureaseinhibitors in soil systems is to mix both urea and the test compoundthroughout the soil and determine the effects of the test compound onrate of urea hydrolysis [L. A. Douglass and J. M. Bremner, Soil Biol.Biochem. 3:309-315 (1971); J. M. Bremner and R. L. Mulvaney, UreaseActivity in Soils, Chapter 5, IN Soil Enzymes p. 149-195, R. G. Burns(Ed.), Academic Press, (1978)].

Test compounds in Example IV, infra, were evaluated using this method asfollows: 100 g of a urease active soil (Hastings silt loam), 40 ml H₂ O,410 mg of urea powder, and 41 mg of powdered inhibitor were mixed wellin a 0.25-liter cylindrical polystyrene container (6×9.8 cm) beforeincubation. The containers were capped and placed in an incubatormaintained at 25° C. After 1, 3, and 6 days, the 100 g soil sample wasextracted with 250 ml of 2M KCl containing 5 ppm of phenylphosphorodiamidate. The KCl-PPDA extracts were analyzed on the AutoAnalyzer II [Technicon method No. 40001 FD4, Technicon, Tarrytown, N.Y.,U.S.A. (1974)].

Test compounds in Examples I, II, III, and V, infra, were evaluated insoil systems by an alternative procedure in which powdered mixtures ofurea and test compounds were applied in narrow bands in the soil ratherthan being mixed throughout the soil. The banded configuration is notonly applicable to banded applications, but also results inconcentration gradients of urea, urea hydrolysis products, testcompounds, and test compound decomposition products similar to those inthe immediate vicinity of urea granules containing test compounds.Another advantage of the banded configuration compared with mixingthroughout the soil is that slightly soluble test compounds can beeasily band applied, whereas it is difficult to achieve a known degreeof mixing of a small quantity of slightly soluble test compound withsoil. The banded configuration also enables testing for ureaseinhibition under realistic soil conditions prior to the development oftechniques for cogranulating a wide range of test compounds with urea.

Specifics of the procedure for evaluating test compounds (Examples I,II, III, and V) were the following. Urease active soil (Hastings siltloam) was moistened to a moisture content of 20 percent (dry weightbasis) and preincubated at room temperature for 2 days. Plexiglascontainers (6×6×6 cm) were one-half filled with soil and packed to abulk density of 1.1 g/cm³. Urea or urea plus inhibitor (thoroughlymixed) was distributed in a narrow band, 6 cm long on the soil surface.The containers were then filled with soil, again packing to a bulkdensity of 1.1 g/cm³. The containers were incubated at 25° C. for thedesired reaction period after which the containers were frozen to about-5° C. to stop urea hydrolysis. Immediately prior to extracting theremaining urea from the soil, said soil was allowed to thaw. Soil fromeach container was thoroughly mixed, and a 10-g sample was extractedwith 100 ml of 2M KCl containing phenylmercuric acetate to prevent ureahydrolysis during handling [ L. A. Douglass and J. M. Bremner, Soil Sci.Soc. Am. Proc. 34:859-862 (1970)]. Urea in the extracts was determinedwith an automated version of the colorimetric procedure [L. A. Douglassand J. M. Bremner, Anal. Letters 3(2):79-87 (1970)].

Test compounds in Example VI, infra, were evaluated in soil systems by amodification of the Douglass and Bremner [L. A. Douglass and J. W.Bremner, Soil Siol. Biochem. 3:309-315 (1971)] procedure in whichsolutions or suspensions of the test compounds were mixed throughout thesoil. Then at selected time intervals, urea was added to thesoil-inhibitor mixtures. The advantage of this procedure is that soilurease inhibition by the test compounds can be detected for time periodslasting up to several weeks or longer. Rapid urea hydrolysis in othertest procedures may limit their applicability to test compounds thathave slow and sustained inhibition properties. As was previouslydemonstrated in FIG. 2 and at least in part by the data in Table VII ofExample VI infra, the test compounds show slow and sustained inhibitoryproperties.

Specifics of the procedure for evaluating the test compounds in ExampleVI were the following. Urease active soil (Crowley silt loam fromLouisiana) was moistened to a moisture content of 16% (dry weight basis)and preincubated at room temperature for 2 days. One milliliter ofsolution or suspension containing 7 micromoles of each test compound wasadded to 120 g of moist soil and mixed well. Polyvinylchloride cylinders12 cm long and 4 cm in diameter were filled with the inhibitor-soilmixture and packed to a bulk density of about 0.9 g/cm³ and covered witha perforated plastic film. These columns of inhibitor-soil mixtures plusuntreated check columns were incubated at 30° C. for periods of 0, 3, 7,14, and 21 days at constant moisture content. At each time interval, 1ml of urea solution containing 50 mg of urea was mixed well with theinhibitor-soil mixture from a single column. The urea-inhibitor-soilmixture was packed in a column to a bulk density of about 0.9 g/cm³,covered with a perforated plastic film, and reincubated for 16 hours at30° C. Following the 16 hours of reincubation, about 120 g of theurea-inhibitor-soil mixture was extracted with 250 ml of 2M KClcontaining 5 ppm of phenylphosphorodiamidate. The KCl-PPDA extracts wereanalyzed on the Auto Analyzer II [Technicon method No. 40001 FD4,Technicon, Tarrytown, N.Y., U.S.A. (1974)]. Three replicates were usedon all treatments and checks.

EXAMPLES

In order that those skilled in the art may better understand how thepresent invention can be practiced, the following examples are given byway of illustration and not necessarily by way of limitation. Names ofcompounds used in the examples and their chemical formulas are shown inTable I below.

                  TABLE I                                                         ______________________________________                                        Compounds and Chemical Formulas                                               Name                  Formula                                                 ______________________________________                                        Phenyl phosphorodiamidate                                                                           (C.sub.6 H.sub.5 O)PO(NH.sub.2).sub.2                   Phosphoryltriamide    PO(NH.sub.2)3                                           Acetohydroxamic acid  CH.sub.3 C(NOH)(OH)                                     Hydroxyurea           CO(NH.sub.2)(NHOH)                                      Ammonium thiocyanate  NH.sub.4 SCN                                            Thiourea              CS(NH.sub.2).sub.2                                      2,2,4,4,6,6-hexaaminocyclotriphosphaza-                                                             P.sub.3 N.sub.3 (NH.sub.2).sub.6                        triene                                                                        2,2,4,4,6,6-hexachlorocyclotriphosphaza-                                                            P.sub.3 N.sub.3 Cl.sub.6                                triene                                                                        2,2,4,4,-tetra(monomethylamino)-6,6-                                                                P.sub.3 N.sub.3 (NH.sub.2).sub.2 (NHCH.sub.3).sub.4                           1                                                       diaminocyclotriphosphazatriene                                                2,2,4,4,6,6-hexa(dimethylamino)cyclotri-                                                            P.sub.3 N.sub.3 [N(CH.sub.3).sub.2 ].sub.6              phosphazatriene                                                               2,2,4,4,6,6-hexa(monomethylamino)cyclotri-                                                          P.sub.3 N.sub.3 (NHCH.sub.3).sub.6                      phosphazatriene                                                               2,2,4,4-tetrachloro-6,6-di(dimethylamino)                                                           P.sub.3 N.sub.3 Cl.sub.4 [N(CH.sub.3).sub.2                                   ].sub.2                                                 cyclotriphosphazatriene                                                       2-phenoxy-2,4,4,6,6-pentaaminocyclotri-                                                             P.sub.3 N.sub.3 (NH.sub.2).sub.5 (OC.sub.6 H.sub.5)     phosphazatriene                                                               2,4-diphenoxy-2,4,6,6-tetraaminocyclotri-                                                           P.sub.3 N.sub.3 (NH.sub.2).sub.4 (OC.sub.6 H.sub.5).                          sub.2                                                   phosphazatriene                                                               2,4,6-triphenoxy-2,4,6-triaminocyclotri-                                                            P.sub.3 N.sub.3 (NH.sub.2).sub.3 (OC.sub.6 H.sub.5).                          sub.3                                                   phosphazatriene                                                               2-phenoxy-2,4,4,6,6-pentachlorocyclotri-                                                            P.sub.3 N.sub.3 Cl.sub.5 (OC.sub.6 H.sub.5)             phosphazatriene                                                               2,4-diphenoxy-2,4,6,6-tetrachlorocyclotri-                                                          P.sub.3 N.sub.3 Cl.sub.4 (OC.sub.6 H.sub. 5).sub.2      phosphazatriene                                                               2,4,6-triphenoxy-2,4,6-trichlorocyclotri-                                                           P.sub.3 N.sub.3 Cl.sub.3 (OC.sub.6 H.sub.5).sub.3       phosphazatriene                                                               ______________________________________                                    

EXAMPLE 1

2,2,4,4,6,6-hexaaminocyclotriphosphazatriene was prepared from2,2,4,4,6,6-hexachlorocyclotriphosphazatriene and anhydrous ammonia bythe procedure of Sowerby and Audrieth [D. B. Sowerby and L. F. Audrieth,Chem. Ber. 94:2670 (1961)]. Its relative effectiveness, versus a numberof other known urease inhibitors, was tested by the following procedure.Urease active soil (Hastings silt loam) was moistened to a moisturecontent of 20 percent and preincubated at room temperature for 2 days.Plexiglas containers (6×6×6 cm) were one-half filled with soil andpacked to a bulk density of 1.1 g/cm³. Urea or urea plus inhibitor(thorougly mixed) was distributed in a narrow band, 6 cm long, on thesurface of the soil. The containers were then filled with soil and againpacked to a bulk density of 1.1 g/cm³. The containers were incubated at25° C. for the desired reaction period. The soil from each container wasthoroughly mixed, and a 10-g sample was extracted with 100 ml of 2M KClcontaining 5 ppm phenylmercuric acetate to prevent urea hydrolysisduring handling [L. A. Douglas and J. M. Bremner, Soil Sci. Soc. Am.Proc. 34:859-862 (1970)]. The urea in the extracts was determinedcolorimetrically as a measure of the unhydrolyzed urea. The results of3-day and 6-day tests for equimolar inhibitor contents are given inTable II below. These results show the2,2,4,4,6,6-hexaaminocyclotriphosphazatriene is comparable to PPDA as aninhibitor of soil urease activity. In 3-day tests, its performanceexceeds that of all other inhibitors tested, except phenylphosphorodiamidate. Its longer term effectiveness (6 days) is evengreater than that of phenyl phosphorodiamidate.

                  TABLE II                                                        ______________________________________                                        Urea Hydrolysis in Bands of Urea as Affected by Various                       Urease Inhibitors.sup.a (Equimolar Basis) in Three Replications               With Hastings Silt Loam Soil at 25° C.                                                         Unhy-                                                                         drolyzed                                                                      Urea, %                                               N     Weight,                Weight,                                                                              3    6                                    Source                                                                              mg/Band  Inhibitor     mg/Band                                                                              Days Days.sup.b                           ______________________________________                                        Urea  .sup. 410.sup.c                                                                        --                    0.4 0.2                                  Urea  410      (C.sub.6 H.sub.5 O)PO(NH.sub.2).sub.2                                                       .sup. 43.sup.d                                                                       68.3 12.9                                 Urea  410      (PO(NH.sub.2).sub.3                                                                         23     43.4 1.8                                  Urea  410      CH.sub.3 C(NOH)(OH)                                                                         19     26.2 6.7                                  Urea  410      CO(NH.sub.2)(NHOH)                                                                          19     20.3 0.2                                  Urea  410      NH.sub.4 SCN  19      3.5 0.5                                  Urea  410      CS(NH.sub.2).sub.2                                                                          19     16.2 0.4                                  Urea  410      P.sub.3 N.sub.3 (NH.sub.2).sub.6                                                            57     48.1 16.8                                 ______________________________________                                         .sup.a The soil urease inhibitory properties of a number of the tested        compounds have also been demonstrated by other investigators:                 Acetohydroxamic acid  [K. B. Pugh and J. S. Waid, Soil. Biol. Biochem.        1:195-206 (1969)].                                                             Hydroxyurea  [W. N. Fishbein, T. S. Winter, and J. D. Davidson, J. Biol.     Chem. 240:2402-2406 (1965)].                                                  Ammonium thiocynate  Unpublished TVA data.                                    Thiourea  [S. S. Malhi and M. Nyborg, Plant and Soil 51:177-186 (1979)].      Phenyl phosphorodiamidate and phosphoryltriamide  [D. A. Martins and J. M     Bremner, Soil Sci. Soc. Am. J. 48:302-305 (1984)].                            .sup.b Urea was completely hydrolyzed in all treatments in 9 days.            .sup.c N rate equivalent to 100 kg/ha applied in bands 30 cm apart. N         source weights are for a band 6 cm long and represent 6.72 moles              urea/band.                                                                    .sup.d Inhibitor weights represent 0.25 millimoles inhibitor/band.       

EXAMPLE II

The results of tests similar in procedure to those utilized in ExampleI, supra, in which 2,2,4,4,6,6-hexaaminocyclotriphosphazatriene,phosphoryltriamide, and phenyl phosphorodiamidate were tested at equalweight contents (10 percent) are shown in Table III below. Again theperformance of 2,2,4,4,6,6-hexaaminocyclotriphosphazatriene exceeds thatof phosphoryltriamide in 3-day tests and is much more effective in 6-daytests.

                  TABLE III                                                       ______________________________________                                        Urea Hydrolysis in Bands of Urea as Affected by Urease                        Inhibitors Applied at a Rate of 10 Percent of Urea (wt/wt Basis);             Four Replications With Hastings Silt Loam Soil at 25° C.                                       Unhy-                                                                         drolyzed                                                                      Urea, %                                               N     Weight,                Weight,                                                                              3                                         Source                                                                              mg/Band  Inhibitor     mg/Band                                                                              Days 6 Days                               ______________________________________                                        Urea   410a    (C.sub.6 H.sub.5 O)PO(NH.sub.2).sub.2                                                       41     69.0 11.8                                 Urea  410      PO(NH.sub.2).sub.3                                                                          41     48.1 1.1                                  Urea  410      P.sub.3 N.sub.3 (NH.sub.2).sub.6                                                            41     49.7 9.5                                  Urea  410      --            --      0.0 0.0                                  ______________________________________                                         a N rate equivalent to 100 kg/ha applied in bands 30 cm apart; N source       weights are for a band 6 cm long.                                        

EXAMPLE III

The tests comprising this example were conducted along the same linesand with similar procedures as outlined in Example I, supra. The resultsof these tests, which are tabulated in Table IV below, show that anumber of derivatives of 2,2,4,4,6,6-hexaaminocyclotriphosphazatriene,wherein some or all of the hydrogens were substituted for by variousgroups, such as halogen and methyl, did not live up to expectations inthat they did not prove to be effective urease inhibitors.

                  TABLE IV                                                        ______________________________________                                        Urea Hydrolysis in Bands of Urea as Affected by Urease Inhibitor              Test Compounds Applied at a Rate of 10 Percent of Urea (wt/wt                 Basis); Two Replications With Hastings Silt Loam Soil                                                  Unhy-                                                                         drolyzed                                                                      Urea, %                                              N     Weight,                 Weight,                                                                              3    6                                   Source                                                                              mg/Band  Inhibitor      mg/Band                                                                              Days Days                                ______________________________________                                        Urea   410a    (C.sub.6 H.sub.5 O)PO(NH.sub.2).sub.2                                                        41     62.6 10.1                                Urea  410      PO(NH.sub.2).sub.3                                                                           41     54.2 2.6                                 Urea  410      P.sub.3 N.sub.3 (NH.sub.2).sub.6                                                             41     53.8 9.5                                 Urea  410      P.sub.3 N.sub.3 Cl.sub.6                                                                     41     3.2  0.0                                 Urea  410      P.sub.3 N.sub.3 (NH.sub.2).sub.2 (NHCH.sub.3).sub.4                                          41     0.5  0.0                                 Urea  410      P.sub.3 N.sub.3 [N(CH.sub.3).sub.2 ].sub.6                                                   41     0.0  0.0                                 Urea  410      P.sub.3 N.sub.3 (NH.sub.2).sub.6 + HCHO                                                      41     3.1  0.0                                 Urea  410      P.sub.3 N.sub.3 (NHCH.sub.3).sub.6                                                           41     0.0  0.0                                 Urea  410      P.sub.3 N.sub.3 Cl.sub.4 [N(CH.sub.3).sub.2                                                  41sub.2                                                                              0.0  0.0                                 Urea  410      --             --     0.9  0.0                                 ______________________________________                                         a N rate equivalent to 100 kg/ha applied in bands 30 cm apart; N source       weights are for a band 6 cm long.                                        

EXAMPLE IV

2-phenoxy-2,4,4,6,6-pentachlorocyclotriphosphazatriene,2,4-diphenoxy-2,4,6,6-tetrachlorocyclotriphosphazatriene, and2,4,6-triphenoxy-2,4,6-trichlorocyclotriphosphazatriene were preparedfrom sodium phenoxide and 2,2,4,4,6,6-hexachlorocyclotriphosphazatrieneby the procedure of Dell, Fitzsimmons, and Shaw [D. Dell, B. W.Fitzsimmons, and R. A. Shaw, J. Chem. Soc., 4070 (1965)]. Thesecompounds were aminated with anhydrous ammonia in a Parr bomb to yieldpure 2-phenoxy-2,4,4,6,6-pentaaminocyclotriphosphazatriene,2,4-diphenoxy-2,4,6,6-tetraaminocyclotriphosphazatriene, and2,4,6-triphenoxy-2,4,6-triaminocyclotriphosphazatriene. The relativeinhibition of the amino compounds versus the known soil ureaseinhibitor, phenyl phosphorodiamidate, was tested by the followingprocedure. Following the procedure of Douglas and Bremner, [L. A.Douglas and J. M. Bremner, Soil Sci. Soc. Am. Proc. 34:859-862 (1970)]urease active soil (Hastings silt loam) was moistened to a moisturecontent of 20 percent and preincubated at room temperature for 2 days.The preincubated soil (100 g) was then well mixed with 20 ml of water,410 mg of urea, or 410 mg of urea plus 41 mg of inhibitor and placed ina cylindrical polystyrene container (6×9.8 cm) and incubated at 25° C.for the desired reaction period. The soil from each container wasthoroughly mixed and a 100-g sample was extracted with 250 ml of 2M KClcontaining 5 ppm of phenyl phosphorodiamidate to prevent urea hydrolysisduring handling. The urea in the extracts was determinedcolorimetrically as a measure of the unhydrolyzed urea. The results of1-day, 3-day, and 6-day tests are given in Table V below. The resultsdemonstrate that 2-phenoxy-2,4,4,6,6-pentaaminocyclotriphosphazatrieneand 2,4-diphenoxy-2,4,6,6-tetraaminocyclotriphosphazatriene have soilurease inhibitory properties comparable to phenyl phosphorodiamidate forthe 1-, 3-, and 6-day tests. The soil urease inhibitory properties of2,4,6-triphenoxy-2,4,6-triaminocyclotriphosphazatriene and2,2,4,4,6,6-hexaaminocyclotriphosphazatriene are less than that ofphenyl phosphorodiamidate in the well-mixed system.

                                      TABLE V                                     __________________________________________________________________________    Urea Remaining Unhydrolyzed in Three Replications in Hastings Silt Loam       Soil                                                                          Incubated at 25° C. in the Prescence of Compounds Tested for           Urease Inhibition in a                                                        Well-Mixed System                                                                                         Unhydrolyzed Urea, %                                                          Well-Mixed                                        N Source                                                                           Weight, mg                                                                          Inhibitor  Weight, mg                                                                          1-Day                                                                             3-Day                                                                             6-Day                                     __________________________________________________________________________    Urea 410   P.sub.3 N.sub.3 (NH.sub.2).sub.5 (OC.sub.6 H.sub.5)                                      41    96  82  44                                        Urea 410   P.sub.3 N.sub.3 (NH.sub.2).sub.4 (OC.sub.6 H.sub.5).sub.2                                41    94  74  36                                        Urea 410   P.sub.3 N.sub.3 (NH.sub.2).sub.3 (OC.sub.6 H.sub.5).sub.3                                41    82  37   0                                        Urea 410   P.sub.3 N.sub.3 (NH.sub.2).sub.6                                                         41    86  57   9                                        Urea 410   (C.sub.6 H.sub.5 O)PO(NH.sub.2).sub.2                                                    41    100 99  46                                        Urea 410   --         --    80  13   0                                        __________________________________________________________________________

EXAMPLE V

2-phenoxy-2,4,4,6,6-pentaaminocyclotriphosphazatriene,2,4-diphenoxy-2,4,6,6-tetraaminocyclotriphosphazatriene, and2,4,6-triphenoxy-2,4,6-triaminocyclotriphosphazatriene were prepared asdescribed in Example IV, supra. The relative urease inhibitorycharacteristics were tested using Hastings silt loam and a banded systemas described in Example I, supra. The results of 3-day and 6-day testsare given in Table VI below.

                  TABLE VI                                                        ______________________________________                                        Urea Remaining Unhydrolyzed in Three Replications in Hastings                 Silt Loam Soil Incubated at 25° C. in the Presence of Compounds        Tested for Urease Inhibition in a Banded System                                                        Unhy-                                                                         drolyzed                                                                      Urea, %                                                                       Banded                                               N     Weight                  Weight 3-   6-                                  Source                                                                              mg/Band  Inhibitor      mg/Band                                                                              Day  Day                                 ______________________________________                                        Urea  410      P.sub.3 N.sub.3 (NH.sub.2).sub.5 (OC.sub.6 H.sub.5)                                          41     61 7                                     Urea  410      P.sub.3 N.sub.3 (NH.sub.2).sub.4 (OC.sub.6 H.sub.5).sub.2                                    41     60 4                                     Urea  410      P.sub.3 N.sub.3 (NH.sub.2).sub.3 (OC.sub.6 H.sub.5).sub.3                                    41     21 0                                     Urea  410      P.sub.3 N.sub.3 (NH.sub.2).sub.6                                                             41     64 8                                     Urea  410      (C.sub.6 H.sub.5 O)PO(NH.sub.2).sub.2                                                        41     82 24                                    Urea  410      --             --     21 0                                     ______________________________________                                    

The results show that2-phenoxy-2,4,4,6,6-pentaaminocyclotriphosphazatriene,2,4-diphenoxy-2,4,6,6-tetraaminocyclotriphosphazatriene, and2,2,4,4,6,6-hexaaminocyclotriphosphazatriene inhibit soil urease duringthe 3-day and 6-day tests.

EXAMPLE VI

2-phenoxy-2,4,4,6,6-pentaaminocyclotriphosphazatriene,2,4-diphenoxy-2,4,6,6-tetraaminocyclotriphosphazatriene,2,4,6-triphenoxy-2,4,6-triaminocyclotriphosphazatriene,2,2,4,4,6,6-hexaaminocyclotriphosphazatriene were prepared as describedin Examples I and IV, phosphoryltriamide and phenyl phosphorodiamidatewere used as reference, supra. The soil urease inhibition was testedusing Crowley silt loam and a well-mixed system modified from that ofDouglass and Bremner [L. A. Douglas and J. M. Bremner, Soil Biol.Biochem. 3:309-315 (1971)]. The results of the tests are given in TableVII below.

                  TABLE VII                                                       ______________________________________                                        Urease Inhibition in Percent in Three Replications                            in Crowley Silt Loam Soil Incubated at 30° C.                          in the Presence of Compounds Tested for Urease Inhibition                     in the Extended Time Evaluation System                                                     Soil Urease Inhibition.sup.a                                                  Days                                                                          0      3     7       14  21                                                   (%)                                                              ______________________________________                                        P.sub.3 N.sub.3 (NH.sub.2).sub.5 (OC.sub.6 H.sub.5)                                          95       75    49    42  44                                    P.sub.3 N.sub.3 (NH.sub.2).sub.4 (OC.sub.6 H.sub.5).sub.2                                    75       94    71    69  65                                    P.sub.3 N.sub.3 (NH.sub.2).sub.3 (OC.sub.6 H.sub.5).sub.3                                    22       79    74    84  82                                    P.sub.3 N.sub.3 (NH.sub.2).sub.6                                                             46       23    20     4   4                                    (C.sub.6 H.sub.5 O)PO(NH.sub.2).sub.2                                                        100      35    27     4   0                                    PO(NH.sub.2).sub.3                                                                           29        4    12     0   0                                    ______________________________________                                         .sup.a L. A. Douglass and J. M. Bremner, Soil Biol. Biochem. 3:309-315        (1971).                                                                  

Specifics of the procedure for evaluating the test compounds in ExampleVI are as follows. Urease active soil (Crowley silt loam from Louisiana)was moistened to a moisture content of 16% (dry weight basis) andpreincubated at room temperature for 2 days. One milliliter of solutionor suspension containing 7 micromoles of each test compound was added to120 g of moist soil and mixed well. Polyvinylchloride cylinders, 12 cmlong and 4 cm in diameter, were filled with the inhibitor-soil mixtureand packed to a bulk density of about 0.9 g/cm³ and covered with aperforated plastic film. These columns of inhibitor-soil mixtures plusuntreated check columns were incubated at 30° C. for periods of 0, 3, 7,14, and 21 days at constant moisture content. At each time interval, 1ml of urea solution containing 50 mg of urea was mixed well with theinhibitor-soil mixture from a single column. The urea-inhibitor soilmixture was packed in a column to a bulk density of about 0.9 g/cm.sup.3, covered with a perforated plastic film, and reincubated for 16 hoursat 30° C. Following the 16 hours of reincubation, about 120 g of theurea-inhibitor-soil mixture was extracted with 250 ml of 2M KClcontaining 5 ppm of phenylphosphorodiamidate. The KCl-PPDA extracts wereanalyzed on the Auto Analyzer II [Technicon method No. 40001 FD4,Technicon, Tarrytown, N.Y., U.S.A. (1974)]. Three replicates were usedon all treatments and checks.

This modification of the inhibitor-soil-urea evaluation procedurepermits the measurement of urease inhibition in a system that isindependent of the time of urea addition. This contrasts with theconventional methods used to collect the data presented in FIG. 1 andTables II to V. The data in Tables II to V in part show that essentiallyall urea in the uninhibited soil tests was hydrolyzed by the soil ureasein 6 days or less. In the modified procedure, supra, the unhydrolyzedurea in the uninhibited soil samples averaged 66%-70% at the time ofanalysis for time periods up to 21 days, thus verifying the advantage ofthis procedure in comparison with the conventional methods.

In FIG. 1 the conventional test shows that none of the test compoundsare effective inhibitors beyond about 10 days. However, when the samecompounds are tested in the time independent modified soil evaluationprocedure, the results (FIG. 2) show that three of the four testcompounds, all phenoxy derivatives, are clearly superior to thereference standard PPDA during the 21-day test period. The fourthcompound--2,2,4,4,6,6-hexaaminocyclotriphosphazatriene--is as good orbetter than PPDA for the time period from 14 to 21 days.

FIG. 2 clearly shows that PPDA demonstrates complete (100%),instantaneous urease inhibition at 0 days, but then shows a rapiddecrease in percent urease inhibition (only 35% inhibition at 3 days and0% inhibition at 21 days). All of the cyclotriphosphazatrienes havelower initial percent urease inhibition than PPDA as well as individualand characteristic inhibition rates. In the case of thehexaaminocyclotriphosphazatriene, its percent inhibition equals orexceeds that of the reference PPDA for periods longer than 14 days andafter 21 days this compound still exhibits some urease inhibition (about4%) while PPDA shows no inhibition. For time periods greater than 3days, the phenoxy-substituted cyclotriphosphazatrienes show highpercentages of urease inhibition (between 75% and 94%) and were able tosustain urease inhibition at levels between 50% and 84% during theperiod from 7 to 21 days. During the early time period (0 to 7 days),[P₃ N₃ (NH₂)₅ (OC₆ H₅)] and [P₃ N₃ (NH₂)₄ (OC₆ H₅)₂ ] maintained highlevels of percent urease inhibition, but their effectiveness declinedslightly during the period between 7 and 21 days. However, during theperiod between 7 and 21 days, [P₃ N₃ (NH₂)₃ (OC₆ H₅)₃ ] was able tomaintain urease inhibition at levels greater than 80%. Thus, it isapparent from FIG. 2 that systematic increases in the degree of phenoxysubstitution changes the pattern of inhibition of thecyclotriphosphazatrienes with the result that a high percentage ofurease inhibition of soil urease activity can be maintained for periodsof at least 21 days.

Thus, only the clear elucidation of the sustained time inhibition ofurease activity by the test compounds by the modified evaluationprocedure makes it possible to understand that physical mixing of thesubstituted cyclotriphosphazatriene derivatives in various proportionswill provide sustained percentage urease inhibition at preselectedlevels and for preselected time periods that can be controlled byvarying the quantity of specific test compounds in the formulation.

To illustrate the use of mixtures of the test compounds to providesustained urease inhibition for extended time periods, the data (inpart) from Table VII in Example VI have been used to calculate theinhibition of some two- and three-component mixtures. These exampleshave been selected for convenience and clarity and similar results couldhave been obtained using other 2-, 3-, and 4-component mixtures toobtain similar levels of percent urease inhibition for extended timeperiods.

The binary mixtures were calculated using two phenoxy-substitutedcyclotriphosphazatrienes [P₃ N₃ (NH₂)₅ (OC₆ H₅)] and [P₃ N₃ (NH₂)₃ (OC₆H₅)₃ ]. All the mixtures shown in FIG. 3 will provide 50% to 80% ureaseinhibition for time periods of 3 days and extending up to at least 21days. Monte Carlo statistical simulations show that a mixture of 50% [P₃N₃ (NH₂)₅ (OC₆ H₅)] and [P₃ N₃ (NH₂)₃ (OC₆ H₅)₃ ] will provide anessentially linear urease inhibition at 65% urease inhibition for thetime period from 0 to 21 days.

Similarly, the tertiary mixtures shown in FIG. 4 were calculated byadding the third phenoxy-substituted cyclotriphosphazatrienes [P₃ N₃(NH₄)₄ (OC₆ H₅)₂ ] to the phenoxy-substituted cyclotriphosphazatrienesused in the binary mixture calculations. The results shown in FIG. 4show that tertiary mixtures will provide 60% to 80% urease inhibitionfor time periods of 3 days and extending to at least 21 days.

The high initial percentage urease inhibition (up to 7 days) resultsfrom [P₃ N₃ (NH₂)₅ (OC₆ H₅)] component of the mixtures while thepercentage of urease inhibition between 7 days and 21 days results fromthe combined effects of [P₃ N₃ (NH₂)₄ (OC₆ H₅)₂ ] and [P₃ N₃ (NH₂)₃ (OC₆H₅)₃ ].

Thus, the combination of the various individual inhibition curves andthe specific desirable inhibition rates will allow the development ofoptimal mixtures of substituted cyclotriphosphazatriene derivatives,especially the phenoxy-substituted derivatives, with specified time andinhibition intensity characteristics to meet the demands of a widevariety of agroclimatic, soil, crop, and management conditions andpractices.

While we have shown and described particular embodiments of the presentinvention, modifications and variations thereof will occur to thoseskilled in the art. We wish it to be understood, therefore, that theappended claims are intended to cover such modifications and variationswhich are within the true scope and spirit of the present invention.

What we claim as new and desire to secure by Letters Patent of theUnited States is:
 1. A method for controlling enzymatic decomposition ofurea juxtaposed soil systems, said enzymatic decomposition of said ureabeing to ammonia and carbonic acid and being due to the action of theenzyme urease thereupon, said method consisting essentially of exposingsaid enzyme to relatively small predetermined amounts of a compound ofthe formula ##STR6## wherein R₁ =R₁ '=R₂ =R₂ '=R₃ =R₃ '=NH₂.
 2. Themethod of claim 1 wherein said compound having the formula shown thereinis applied with said urea in juxtaposition with said soil system at therate ranging from about 0.01 percent to about 10 percent of urea wt/wtbasis.
 3. A method for controlling enzymatic decomposition of ureajuxtaposed soil systems, said enzymatic decomposition of said urea beingto ammonia and carbonic acid and being due to the action of the enzymeurease thereupon, said method consisting essentially of exposing saidenzyme to relatively small predetermined amounts of a compound of theformula ##STR7## wherein R₁ '=R₂ '=R₃ '=R₂ =R₃ =NH₂ and R₁ =OC₆ H₅. 4.The method of claim 3 wherein said dcompound having the formula showntherein is applied with said urea in juxtaposition with said soil systemat the rate ranging from about 0.01 percent to about 10 percent of ureawt/wt basis.
 5. A method for controlling enzymatic decomposition of ureajuxtaposed soil systems, said enzymatic decomposition of said urea beingto ammonia and carbonic acid and being due to the action of the enzymeurease thereupon, said method consisting essentially of exposing saidenzyme to relatively small predetermined amounts of a compound of theformula ##STR8## wherein R₁ '=R₂ '=R₃ '=R₃ =NH₂ and R₁ =R₂ =OC₆ H₅. 6.The method of claim 5 wherein said compound having the formula showntherein is applied with said urea in juxtaposition with said soil systemat the rate ranging from about 0.01 percent to about 10 percent of ureawt/wt basis.
 7. A method for controlling enzymatic decomposition of ureajuxtaposed soil systems, said enzymatic decomposition of said urea beingto ammonia and carbonic acid and being due to the action of the enzymeurease thereupon, said method consisting essentially of exposing saidenzyme to relatively small predetermined amounts of a compound of theformula ##STR9## wherein R₁ '=R₂ '=R₃ '=NH₂ and R₁ =R₂ =R₃ =OC₆ H₅. 8.The method of claim 7 wherein said compound having the formula showntherein is applied with said urea in juxtaposition with said soil systemat the rate ranging from about 0.01 percent to about 10 percent of ureawt/wt basis.
 9. A method for controlling enzymatic decomposition of ureajuxtaposed soil systems, said enzymatic decomposition of said urea beingto ammonia and carbonic acid and being due to the action of the enzymeurease thereupon, said method consisting essentially of exposing saidenzyme to relatively small predetermined amounts of compounds of theformula ##STR10## wherein R₁ '=R₂ '=R₃ '=NH₂ ; R₁ =OC₆ H₅ ; and R₂ andR₃ are selected from the group consisting of NH₂, OC₆ H₅, and mixturesthereof.
 10. The method of claim 9 wherein said compounds having theformulae shown therein are applied with said urea in juxtaposition withsaid soil system at the rate ranging from about 0.01 percent to about 10percent of urea wt/wt basis.
 11. A method for substantially substainingthe control of enzymatic decomposition of urea juxtaposed soil systems,said enzymatic decomposition of said urea being to ammonia and carbonicacid and being due to the action of the enzyme urease thereupon, saidmethod consisting essentially of exposing said enzyme to relativelysmall predetermined amounts of compounds of the formula ##STR11##wherein R₁ '=R₂ '=R₃ '=NH₂ ; R₁ =OC₆ H₅ ; and R₂ and R₃ are selectedfrom the group consisting of NH₂, OC₆ H₅, and mixtures thereof.
 12. Themethod of claim 11 wherein said compounds having the formulae showntherein are applied with said urea in juxtaposition with said soilsystem at the rate ranging from about 0.01 percent to about 10 percentof urea wt/wt basis to thereby effect a sustained control of saidenzymatic decomposition of said urea of at least about 50 percent forthe period of time ranging from about 3 days to about 21 days subsequentto said application of said compounds to said soil system.
 13. Themethod of claim 12 wherein said compounds comprise from about 25 percentto about 75 percent of the compound of claim 3 and the remainder thereofas the compound of claim
 7. 14. The method of claim 13 wherein saidcompounds comprise about 50 percent of the compound of claim 3 tothereby effect said sustained control at a level of at least about 60percent.
 15. The method of claim 13 wherein said compounds compriseabout 25 percent of the compound of claim 3 to thereby effect saidsustained control at a level of at least about 70 percent.
 16. Themethod of claim 12 wherein said compounds comprise from about 5 percentto about 90 percent of each of the compounds of claims 3, 5, and
 7. 17.The method of claim 16 wherein each of said compounds is present inabout equal portions to thereby effect said sustained control at a levelof at least about 65 percent.
 18. The method of claim 16 wherein saidcompounds of claims 3, 5, and 7 are present therein in portions of about10 percent, about 30 percent, and about 60 percent, respectively, tothereby effect said sustained control at a level of at least about 70percent.
 19. The method of claim 16 wherein said compounds of claims 3,5, and 7 are present therein in portions of about 5 percent, about 5percent, and about 90 percent, respectively, to thereby effect saidsustained control at a level of at least about 75 percent.